Advances in the study of molecules have been led, in part, by improvement in technologies used to characterise the molecules or their biological reactions. In particular, the study of nucleic acids, such as DNA and RNA, and other large biological molecules, such as proteins, has benefited from developing technologies used for sequence analysis and the study of hybridisation events.
An example of the technologies that have improved the study of nucleic acids is the development of fabricated arrays of immobilised nucleic acids. These arrays typically consist of a high-density matrix of polynucleotides immobilised onto a solid support material. Fodor et al., Trends in Biotechnology (1994) 12:19-26, describe ways of assembling the nucleic acid arrays using a chemically sensitised glass surface protected by a mask, but exposed at defined areas to allow attachment of suitably modified nucleotides. Typically, these arrays may be described as “many molecule” arrays, as distinct regions are formed on the solid support comprising a high density of one specific type of polynucleotide.
An alternative approach is described by Schena et al., Science (1995) 270:467-470, where samples of DNA are positioned at predetermined sites on a glass microscope slide by robotic micropipetting techniques.
A further development in array technology is the attachment of the polynucleotides to a solid support material to form single molecule arrays (SMAs). Arrays of this type are disclosed in WO00/06770. The advantage of these arrays is that reactions can be monitored at the single molecule level and information on large numbers of single molecules can be collated from a single reaction.
Although these arrays offer particular advantages in sequencing experiments, the preparation of arrays at the single molecule level is more difficult than at the multi-molecule level, where losses of target polynucleotide can be tolerated due to the multiplicity of the array. There is, therefore, a constant need for improvements in the preparation of single molecule arrays for sequencing procedures. In particular, it is desirable to be able to attach sample polynucleotide (e.g. DNA) from solution under conditions which minimise the non-specific association of sample polynucleotide (e.g. DNA) to the solid support.
Sequencing polynucleotides on a solid support can be difficult because the polynucleotide to be sequenced is typically bound to the solid support indirectly by way of the formation of a hybrid with a support-bound complement. Conditions used in the sequencing protocol can result in disruption to the bonds formed on hybridisation and the target polynucleotide may be removed from the array. By “target polynucleotides” or “target nucleic acid” is meant herein the polynucleotide whose sequence it is desired to determine.
Accordingly, research has been directed to develop sequencing methodologies where the target nucleic acid is bound to a solid support and which address the disruption of polynucleotide duplexes caused by the lability of the hydrogen bonds formed between complementary nucleotide bases. Such techniques have led to the development and use of polynucleotides having hairpin stem-loop structure, referred to hereinafter as hairpin polynucleotides.
The term “hairpin loop structure” refers to a molecular stem and loop formed from the hybridisation of complementary polynucleotides that are covalently linked at one end. The stem comprises the hybridised polynucleotides and the loop is the region that links the two complementary polynucleotides.
WO98/20019 discloses compositions and methods for the preparation of nucleic acid arrays. The general disclosure relates to the preparation of high density multi-molecule arrays, achieved by immobilising polynucleotides on microscopic beads attached to a solid support. Many different uses are proposed for the arrays.
WO97/08183 relates to nucleic acid capture molecules. Hairpin polynucleotide structures are disclosed as being useful as capture molecules in hybridisation-based nucleic acid detection methods.
Hairpin polynucleotides permit improved sequence analysis procedures to be conducted, since a target polynucleotide may be maintained in spatial relationship to a primer. Maintenance of the spatial relationship is made possible not only by the hydrogen bonds formed on hybridisation, but also by the tethering of a known primer to the target polynucleotide, the tether being the “loop” (see WO97/04131).
In WO97/04131, the hairpin is immobilised on a glass support by reaction between a pendant epoxide group on the glass with an internal amino group held within the loop. This method of immobilising hairpin polynucleotides on solid supports is but one of a number of linking methodologies which have been developed to date.
Zhao et al (Nucleic Acids Research, 2001, 29(4), 955-959) disclose the formation of a hairpin polynucleotide which contains multiple phosphorothioate moieties in the loop. The moieties are used to anchor, in more than one position, the hairpin DNA to glass slides pre-activated with bromoacetamidopropylsilane. This chemistry was found to improve attachment of hairpin DNA to glass slides.
The work of Zhao developed upon earlier work of Pirrung et al (Langmuir, 2000, 16, 2185-2191) in which the authors report that 5′-thiophosphate-terminating oligonucleotides could be attached to glass, pre-activated with mono- and dialkoxylated silanes and bromoacetamide.
Phosphorothioate coupling chemistry works well where the solution applied is dried down onto the support. However, the conditions under which phosphorothioate coupling is effected are not applicable in to the preparation of SMAs. This is because when drying down the applied solution in the protocol used for phosphorothioate coupling, this may take place non-uniformly. This is the case when oligonucleotides are spotted onto preactivated glass, for example as taught by Zhao (infra) where small volumes (0.7 nl) are used. Accordingly, clustering can take place on the surface of the support which is clearly undesirable in the preparation of a SMA.